Autophagy, an evolutionarily conserved intracellular lysosomal degradative pathway, is constitutively active in the myocardium, and removes damaged organelles and proteins to ensure cardiomyocyte survival. Rapid upregulation of autophagy is essential for maintaining cardiomyocyte viability under stress, such as during starvation and ischemia, by generating nutrients as a source of energy and removing potentially deleterious proteins and organelles. Autophagy is also critical for removal of abnormal desmin aggregates that cause cardiomyopathy with the R120G mutation in chaperone protein, B crystallin. It is therefore paradoxical that an upregulation of autophagy has been implicated in causing cardiomyocyte death in reperfusion injury, following an ischemic insult. With reperfusion, we have observed an impairment in autophagosome processing, triggered by a burst in reactive oxygen species generation, as a cause of autophagosome accumulation; which implies that flux through autophagy is impaired with ischemia-reperfusion injury. Indeed, we have demonstrated that autophagosome formation is induced in cardiomyocytes by expression of BNIP3, a pro-death protein that is transcriptionally induced by hypoxia and mediates cardiomyocyte death in ischemia- reperfusion injury; but autophagosome processing is impaired as the autophagy-lysosome machinery gets overwhelmed and autophagosomes enclosing damaged mitochondria accumulate. Exogenous expression of transcription factor EB (TFEB), a master regulator of autophagy-lysosome pathway biogenesis, re-established autophagosome processing, with removal of BNIP3-damaged mitochondria and attenuated cardiomyocyte death. We have also observed that starvation, a potent inducer of cardiomyocyte autophagy, provokes transcriptional upregulation of multiple components of the autophagy-lysosome machinery in the mouse heart; and repetitive starvation by intermittently fasting (depriving mice of food for 24 hours every othe day for 6 weeks) results in protection against ischemia-reperfusion (IR) injury, with >50% reduction in infarct size as compared with ad-lib fed mice. This is associated with a rapid fasting-induced nuclear translocation of TFEB from the cytoplasm, suggesting that adequate priming of the autophagy-lysosome machinery by TFEB promotes sufficient autophagy, which is then beneficial in the setting of ischemia-reperfusion injury. In this proposal, we will test the hypothesis that intermittent fasting and exogenous expression of TFEB protect against ischemia-reperfusion induced cardiomyocyte death and protein-aggregate-induced cardiomyopathy and heart failure, by enhancing beneficial autophagy. In specific aim 1, studies will be performed to determine the role of autophagy in intermittent fasting-induced cardioprotection against post-infarction remodeling, by subjecting mice deficient for LAMP2, (which are also autophagy-deficient as LAMP2 is critical for autophagosome-lysosome fusion) to IR injury followed by serial echocardiography and terminal invasive pressure-volume loop studies, to assess changes in left ventricular size and function and development of heart failure, as a function of LAMP2 expression. In specific aim 2, mice with conditional adult onset cardiomyocyte specific overexpression of TFEB will be subjected to IR injury to determine whether exogenous TFEB confers protection against cell death, post-infarction remodeling and heart failure, by enhancing autophagy. In specific aim 3, mice with cardiomyocyte-specific expression of mutant crystallin will be subjected to intermittent fasting or crossed with TFEB overexpressors, to determine whether induction of autophagy prevents protein- aggregate induced heart failure. These studies will evaluate the paradigm that transcriptional induction of autophagy-lysosome machinery enhances stress-induced autophagy, and protects against cardiomyocyte death and heart failure in ischemia-reperfusion injury and desmin-induced cardiomyopathy.